Perfect coordination of the extent and timing of cellular proliferation with terminal differentiation is a genetically controlled process, fundamental for the development of all tissues and organ systems. Deregulations of this tightly regulated process can cause human birth defects and neoplastic transformation. Pbx1 is a homeodomain protein that collaboratively binds DNA with Hox proteins to modulate their DNA binding specificities and is a homolog of Drosophila extradenticle (EXD), whose function in patterning the fly body plan has been demonstrated genetically. Our ongoing efforts and long-term goals utilize genetically modified mouse models to assess the contributions of the Pbx family of Hox cofactors to mammalian patterning and morphogenesis. In embryos, the lack of Pbx1 (Pbx1-l-) results in late gestational lethality, widespread patterning defects of the axial and appendicular skeleton, homeotic transformation of second branchial arch neural crest cell-derived skeletal structures, markedly diminished chondrocyte proliferation, accompanied by precocious chondrocyte hypertrophy, and premature ossification of bone. Unlike Pbx1-l-, both Pbx2 -l-and Pbx3-l- mice do not display gross abnormalities either in patterning or in skeletal development/maturation. Nonetheless, Pbx1-l-; Pbx2-l- mutants die earlier in utero and show drastic exacerbation of the skeletal defects. The goal of this proposal is to finely dissect the genetic control of patterning and skeletal development by the homeobox gene Pbx1, through the following specific aims: 1) genetically uncouple the early roles of Pbxl in patterning from its later roles in cartilage proliferation, differentiation and endochondral ossification, through the generation of knockout mice where Pbxl is inactivated in a tissue-specific manner in neural crest and chondrocytes, by utilizing available Cre mice; 2) characterize the role of Pbx1 in chondrocyte proliferation by using genetically modified (Pbx1-l-) mesenchymal cells in culture, such as Mouse Embryonic Fibroblasts (MEFs), which show a striking growth defect, and Micromass Mesenchyme Cultures; 3) identify unique and overlapping functions of Pbx1 with the related family member Pbx2 in skeletal development and also exclusively in the genetic control of chondrogenesis. Completion of these studies will advance our understanding of the genetic regulation of patterning and skeletal development by Pbx1. Under a broader perspective, this work will shed light on the dramatic effects of the perturbations of skeletal development and hopefully impact on our understanding of the pathogenesis of human birth defects that affect the development of the craniofacial, axial and appendicular skeleton.